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Methods and compositions for selective regulation of protein expression / Monsanto Technology Llc




Methods and compositions for selective regulation of protein expression


The invention provides novel recombinant DNA molecules, compositions, and methods for selectively regulating the expression of a transcribable polynucleotide molecule or recombinant protein in a male reproductive tissue of a transgenic plant. The invention also provides transgenic plants, plant cells, plant parts, seeds, and commodity products comprising such DNA molecules and compositions.



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USPTO Applicaton #: #20170029841
Inventors: Jintai Huang, Youlin Qi, Heping Yang, Yuanji Zhang


The Patent Description & Claims data below is from USPTO Patent Application 20170029841, Methods and compositions for selective regulation of protein expression.


CROSS-REFERENCE TO RELATED APPLICATIONS

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This application claims the benefit of U.S. Provisional Application No. 62/195,546, filed on Jul. 22, 2015, herein incorporated by reference in its entirety.

INCORPORATION OF SEQUENCE LISTINGS

The sequence listing that is contained in the file named “MON5392US_ST25.txt”, which is 26.3 kilobytes (measured in operating system MS-Windows), created on Jun. 28, 2016, is filed herewith and incorporated herein by reference.

BACKGROUND

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OF THE INVENTION Field of the Invention

The invention relates generally to the fields of agriculture, plant breeding, and molecular biology. More specifically, the invention relates to methods and compositions for selectively regulating protein expression in the male reproductive tissue of transgenic plants and uses thereof.

Description of Related Art

Hybrid seed is produced by hybridization or cross-fertilization of closely related plants and can be grown into progeny hybrid plants possessing a desirable combination of traits not possessed by either parent plant. Hybrid plants can display superior agronomic characteristics such as improvement of plant size, yield, nutritional composition, disease resistance, herbicide tolerance, stress tolerance, climatic adaptation, and other desirable traits. Efficient hybrid seed production requires that a plant's own pollen not be permitted to self-fertilize the plant. A major limitation in the production of hybrid seed for many crops is the lack of simple, reliable, and economical methods of making plants male-sterile and incapable of self-fertilization.

In hybrid seed production, pollen production and pollen shed may be prevented in a female parent plant in order to facilitate cross-pollination of the female rather than self-pollination. Such prevention may be achieved by, for example, manual removal of the pollen-containing structures (for example, by manual or mechanical detasseling in maize), use of a genetic means of pollination control (for example, by using cytoplasmic male-sterile or nuclear male-sterile technology), use of a chemical agent, or any combination of these. This can be a labor-intensive and therefore expensive process. In maize, for example, detasseling is typically done in two steps: machine detasseling followed by manual detasseling. Commercial production of hybrid seed using solely chemical gametocides is limited primarily by their general lack of selectivity for gametes and their effect on the other parts of the plant. Thus, methods for improving the efficiency of hybrid seed production are highly desirable.

BRIEF

SUMMARY

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OF THE INVENTION

The invention relates generally to improvements to methods of selectively regulating protein expression in the male reproductive tissue of transgenic plants, recombinant DNA molecules useful in such methods, as well as transgenic plants, cells, and seeds containing such recombinant DNA molecules. The invention provides an improvement over the art by providing male tissue-specific siRNA (mts-siRNA) target elements capable of providing improved selective regulation of the expression of a protein encoded by a transcribable polynucleotide molecule and provides recombinant DNA molecules and compositions comprising such mts-siRNA target elements and methods of using such mts-siRNA target elements for inducing male sterility in transgenic plants for the production of hybrid seed.

In one aspect, the invention provides a recombinant DNA molecule comprising a mts-siRNA target element operably linked to a heterologous transcribable polynucleotide molecule. In one embodiment, the mts-siRNA target element is included within a 3′ untranslated region operably linked to the heterologous transcribable polynucleotide molecule. In another embodiment, the mts-siRNA target element is located between the heterologous transcribable polynucleotide molecule and an operably linked polyadenylation sequence that is part of a 3′ untranslated region. In one embodiment, the mts-siRNA target element comprises a sequence selected from the group consisting of SEQ ID NO: 1-16, 23-92, and complements thereof. In another embodiment, the heterologous transcribable polynucleotide molecule confers herbicide tolerance, for instance vegetative herbicide tolerance, to a plant. In a further embodiment, the heterologous transcribable polynucleotide molecule does not confer male reproductive herbicide tolerance to a plant. In another embodiment, the heterologous transcribable polynucleotide molecule is a glyphosate-tolerant 5-enolypyruvyl shikimate 3-phosphate synthase (EPSPS).

In another aspect, the invention provides a recombinant DNA construct comprising a mts-siRNA target element of the invention operably linked to a heterologous transcribable polynucleotide molecule.

In further aspect, the invention provides a method of producing a recombinant DNA molecule comprising operably linking at least one mts-siRNA target element to a heterologous transcribable polynucleotide molecule. In one embodiment, the mts-siRNA target element comprises a sequence selected from the group consisting of SEQ ID NO: 1-16, 23-92, and complements thereof.

In another aspect, the invention provides a transgenic plant comprising a mts-siRNA target element of the invention. In one embodiment, the transgenic plant comprises the mts-siRNA target element operably linked to a heterologous transcribable polynucleotide molecule. In a further embodiment, the mts-siRNA target element comprises a sequence selected from the group consisting of SEQ ID NO: 1-16, 23-92, and complements thereof. In yet another embodiment, the transgenic plant is produced by transforming a plant with a recombinant DNA molecule or DNA construct comprising at least one mts-siRNA target element operably linked to a heterologous transcribable polynucleotide molecule. In a further aspect, the invention provides a seed, cell, or part of such a transgenic plant. In one embodiment, the plant is a monocotyledonous plant. In another embodiment, the plant is a maize (Zea mays) plant.

In a further aspect, the invention also provides a method of selectively regulating the expression of a protein in a male reproductive tissue of a transgenic plant by expressing in the transgenic plant a recombinant DNA molecule that comprises a mts-siRNA target element operably linked to a heterologous transcribable polynucleotide molecule. In one embodiment, the mts-siRNA target element comprises a sequence selected from the group consisting of SEQ ID NO: 1-16, 23-92, and complements thereof. In another embodiment, the heterologous transcribable polynucleotide molecule confers herbicide tolerance, for instance vegetative herbicide tolerance, to a plant. In a further embodiment, the heterologous transcribable polynucleotide molecule does not confer male reproductive herbicide tolerance to a plant. In another embodiment, the heterologous transcribable polynucleotide molecule is a glyphosate-tolerant EPSPS.

In yet another aspect, the invention provides a method of inducing male sterility in a transgenic plant, including the step of applying herbicide to a transgenic plant that has in its genome a recombinant DNA molecule comprising a mts-siRNA target element operably linked to a heterologous transcribable polynucleotide molecule that confers tolerance to at least a first herbicide to the transgenic plant, wherein the herbicide is applied prior to or concurrently with the development of the male reproductive tissue of the transgenic plant, thereby inducing male-sterility in the transgenic plant. In one embodiment, the heterologous transcribable polynucleotide molecule confers vegetative herbicide tolerance, but does not confer male reproductive herbicide tolerance to the transgenic plant. In another embodiment, the transgenic plant is a maize plant. In a further embodiment, the herbicide application prevents at least pollen development, pollen shed, or anther extrusion in the treated transgenic plant. In another embodiment, the developmental stage of the male reproductive tissue during which herbicide is applied is a stage selected from the group consisting of the V4, V5, V6, V7, V8, V9, V10, V11, V12, V13, and V14 stage of maize plant development. In another embodiment, the herbicide is selected from the group consisting of acetyl coenzyme A carboxylase (ACCase) inhibitors, acetolactate synthase (ALS) inhibitors, photosystem II (PSII) inhibitors, protoporphyrinogen oxidase (PPO) inhibitors, 4-hydroxyphenyl dioxygenase (HPPD) inhibitors, 5-enolypyruvyl shikimate 3-phosphate synthase (EPSPS) inhibitors, glutamine synthetase (GS) inhibitors, and synthetic auxins. In another embodiment, the herbicide is glyphosate and the heterologous transcribable polynucleotide encodes a glyphosate-tolerant EPSPS.

In one aspect, the invention also provides a method of producing hybrid seed comprising applying an effective amount of an herbicide to a transgenic plant comprising in its genome a recombinant DNA molecule comprising a mts-siRNA target element operably linked to a heterologous transcribable polynucleotide molecule, wherein the herbicide is applied prior to or concurrently with the development of the male reproductive tissue of the transgenic plant, thereby inducing male sterility in the transgenic plant; fertilizing the transgenic plant with pollen from a second plant; and allowing hybrid seed to form from the transgenic plant. In one embodiment, the transgenic plant is maize. In another embodiment, the herbicide is glyphosate and the heterologous transcribable polynucleotide molecule is a glyphosate-tolerant EPSPS. In another embodiment, the glyphosate is applied concurrently with development at an effective amount of about 0.125 pounds acid equivalent per acre to about 8 pounds acid equivalent per acre. In another aspect, the invention provides hybrid seed produced by such a method. In one embodiment, the hybrid seed comprises the recombinant DNA molecule.

Other specific embodiments of the invention are disclosed in the following detailed description. Throughout this specification and the claims, unless the context requires otherwise, the word “comprise” and its variations, such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer, element, or step or group of integers, elements, or steps, but not the exclusion of any other integer, element, or step or group of integers, elements, or steps.

BRIEF DESCRIPTION OF THE DRAWINGS

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FIG. 1. Illustration of tassel developmental stages V7 at T2, V8/V9 at T4, V10/V11 at T5, V12 at T6, and VT at T7 showing tassel size and morphology with the lower panel photographs of cross-sections of anthers showing the pollen developmental stage. The stages previously (V10/V11 and V12) used in the art for isolating small RNA molecules for identification of mts-siRNA molecules are indicated by a box with solid line; the stages (V7, V8/V9, V10/V11 and V12) described herein for isolating small RNA molecules are indicated by a box with dashed line.

FIG. 2. Graphical representation of alignment of mts-siRNA sequences on the cDNA sequence provided as SEQ ID NO: 17. The cDNA sequence is indicated from nucleotide 1 to 1826, and the short lines represent the alignment of the complementary strand of individual mts-siRNA sequences, or stretches of adjacent mts-siRNA sequences or overlapping mts-siRNA sequences (relatively longer lines) to the cDNA sequence. The mts-siRNA sequences which are the same strand as the cDNA are not shown. The normalized relative expression level of mts-siRNA is indicated on the left. The boxed area represents a region of the cDNA sequence rich in mts-siRNA targets.

FIG. 3. Diagram illustrating double stranded mts-siRNA, single stranded mts-siRNA, mts-siRNA target sequence within a mRNA, and a region of mRNA with a high number of mts-siRNA target sequences useful as a mts-siRNA target element.

FIG. 4. Photograph of R0 maize tassel and pollen stained with Alexandar stain from double copy transgenic plants containing a recombinant DNA molecule comprising a transgene encoding a glyphosate-tolerant EPSPS protein operably linked to a mts-siRNA target element (SEQ ID NO: 2). Events 1 through 4 were sprayed with 0.75 lb ae/acre glyphosate herbicide at V5 followed by V8 stage and showed tassels with complete male sterility as defined by no anthesis and either nonviable pollen grains or no pollen grains detected in the anthers. Control plants did not receive a glyphosate application and demonstrated normal anthesis and pollen shedding and the microscopic observation detected normal pollen grains.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1—A mts-siRNA target element sequence having 95% sequence identity to nucleotide positions 1429 to 1628 of the cDNA sequence provided herein as SEQ ID NO: 17.

SEQ ID NO: 2—A mts-siRNA target element sequence having 95% sequence identity to nucleotide positions 1429 to 1628 of the cDNA sequence provided herein as SEQ ID NO: 17 and having a single nucleotide change (T69A) relative to SEQ ID NO: 1.

SEQ ID NO: 3—A mts-siRNA target element sequence that corresponds to nucleotide positions 239 to 433 of the cDNA sequence provided herein as SEQ ID NO: 18.

SEQ ID NO: 4—A mts-siRNA target element sequence that corresponds to nucleotide positions 477 to 697 of the cDNA sequence provided herein as SEQ ID NO: 18.




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stats Patent Info
Application #
US 20170029841 A1
Publish Date
02/02/2017
Document #
File Date
12/31/1969
USPTO Class
Other USPTO Classes
International Class
/
Drawings
0


Cells Dna Molecule Dna Molecules Elective Nucleotide Polynucleotide Protein Expression Recombinant Recombinant Dna Molecules Recombinant Protein Transgenic Transgenic Plants

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Monsanto Technology Llc


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20170202|20170029841|methods and compositions for selective regulation of protein expression|The invention provides novel recombinant DNA molecules, compositions, and methods for selectively regulating the expression of a transcribable polynucleotide molecule or recombinant protein in a male reproductive tissue of a transgenic plant. The invention also provides transgenic plants, plant cells, plant parts, seeds, and commodity products comprising such DNA molecules |Monsanto-Technology-Llc
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